Monoclonal Antibodies Against Mature Interleukin-18 Ameliorate Colitis and Repair Goblet Cell Function.

BACKGROUND: Numerous biological interventions and small molecules are used to treat Crohn's disease; however, the effectiveness of these treatments varies largely. Non-responsiveness to biological therapies is associated with interleukin (IL)-18 gene polymorphisms and high IL-18 expression has been implicated in the pathogenesis of Crohn's disease. AIMS: The aim of this study was to elucidate the expression of precursor and mature IL-18 in patients with Crohn's disease who exhibited varied responses to cytokine-targeted treatments and determine whether selective inhibition of mature IL-18 offers a novel therapeutic avenue. METHODS: We generated a monoclonal antibody that specifically recognizes the neoepitope of caspase-cleaved mature IL-18. Expression of precursor and mature IL-18 was analyzed in patients with Crohn's disease. Anti-mature IL-18 monoclonal antibodies were intraperitoneally administered in an acute colitis mouse model, and the disease activity index, body weight loss, tissue pathology, proinflammatory cytokine expression, goblet cell function, and microbiota composition were assessed. RESULTS: Precursor and mature IL-18 expression was upregulated and goblet cell function was impaired in patients with Crohn's disease who were unresponsive to biological therapies. Administration of anti-mature IL-18 antibodies ameliorated induced colitis by repairing goblet cell function and restoring the mucus layer. CONCLUSIONS: The newly developed monoclonal antibody holds promise as a therapeutic alternative for Crohn's disease.

Generation of antagonistic monoclonal antibodies against the neoepitope of active mouse interleukin (IL)-18 cleaved by inflammatory caspases.

Interleukin 18 (IL-18) is a member of the IL-1 family and plays an important role in both the innate and acquired immune systems. It is constitutively expressed as an inactive precursor (24 kDa) in various cell types, and the mature IL-18 (18 kDa) cleaved by inflammatory caspase-1/4 binds to the interleukin-18 receptor, thereby activating downstream signaling pathways. We previously generated anti-human IL-18 antibodies that specifically recognize the human IL-18 neoepitope cleaved by inflammatory caspase-1/4. Because the N-terminal amino acid sequences of the neoepitopes are different between human IL-18 and mouse IL-18, the anti-human IL-18 neoepitope antibodies do not recognize mouse mature IL-18. We have now generated novel anti-mouse IL-18 neoepitope antibodies. We also confirmed CXCL2 secretion from P-815 mouse cells by mouse IL-18 stimulation, and established a simple assay to evaluate the activity of mouse IL-18. Using this evaluation system, we confirmed that the anti-mouse IL-18 neoepitope antibodies could inhibit mouse IL-18. By demonstrating the therapeutic efficacy of the anti-mouse IL-18 neoepitope and function-blocking mAbs established in the present study in mouse models, corresponding to human inflammatory diseases in which IL-18 may be involved, such as inflammatory bowel diseases, we can provide the proof-of-concept that the previously established anti-human IL-18 neoepitope and function-blocking mAbs work in human inflammatory disorders corresponding to mouse models.

Screening of a Panel of Low Molecular Weight Compounds That Inhibit Synovial Fibroblast Invasion in Rheumatoid Arthritis.

Increased invasion of synovial fibroblasts and their involvement in cartilage damage are characteristic phenotypes of rheumatoid arthritis (RA). To identify low molecular weight compounds that suppress synovial fibroblast invasion, a panel of inhibitors (n = 330) was initially screened using a real-time cell analysis system for human synovial fibroblasts that were enzymatically isolated from surgical samples of RA patients. To evaluate the effects of the inhibitors identified in the screen, synovial fibroblast migration was measured using a wound-healing assay, and phosphorylation of intracellular signaling molecules was determined by immunoblots. Several candidate inhibitors were identified in the screen, including inhibitors against platelet-derived growth factor receptor (PDGFR), Akt, PI3K, and glycogen kinase synthetase 3 (GSK-3). These inhibitors strongly suppressed synovial fibroblast migration after 72 h and downregulated phosphorylation of Akt (Ser473) at 48 h. When the inhibitors were removed from the culture conditions, both migration and phosphorylated Akt (Ser473) levels were restored. Furthermore, all the categories of inhibitors except for PDGFR inhibitor IV decreased cell proliferation as well as IL-6 production in synovial fibroblasts. Interestingly, GSK-3 inhibitors increased anti-inflammatory cytokine IL-10 production but suppressed IL-23 production from LPS-primed macrophages obtained from healthy donors. In conclusion, blocking PDGFR, PI3K, or GSK-3 could have therapeutic value as an RA treatment that targets the invasion/migration of synovial fibroblasts.

p53/Mieap-regulated mitochondrial quality control plays an important role as a tumor suppressor in gastric and esophageal cancers.

Mitochondria-eating protein (Mieap) plays a critical role in mitochondrial quality control (MQC) and functions as a p53-inducible tumor suppressor. This study aimed to examine its role in gastric cancer (GC) and esophageal cancer (EC). GC cells were infected with Mieap-overexpressing adenovirus (Ad-Mieap) and subjected to fluorescence-activated cell sorting (FACS), western blotting, and caspase assays. Thereafter, we evaluated the potential disruption of the p53/Mieap-regulated MQC pathway in vivo. Methylation-specific PCR (MSP) for Mieap, NIX, and BNIP3 promoters was performed and p53 mutations were detected using cryopreserved surgical specimens. Exogenous Mieap in GC cells induced the formation of vacuole-like structures (called MIVs, Mieap-induced vacuoles) and caspase-dependent cell death, with the activation of both caspase-3 and caspase-9. Of the 47 GC patients, promoter methylation in Mieap, BNIP3, and NIX was identified in two (4.3%), 29 (61.7%), and zero (0%) specimens, respectively. In total, 33 GC patients (70.2%) inactivated this MQC pathway. Amazingly, BNIP3 promoter in the normal epithelium was highly methylated in 18 of the 47 GC patients (38.3%). In EC patients, this MQC pathway was also inactivated in ten of 12 patients (83.3%). These results indicate that p53/Mieap-regulated MQC plays an important role in upper gastrointestinal (GI) tumor suppression, possibly, in part, through the mitochondrial apoptotic pathway.

Generation and characterization of antagonistic anti-human interleukin (IL)-18 monoclonal antibodies with high affinity: Two types of monoclonal antibodies against full-length IL-18 and the neoepitope of inflammatory caspase-cleaved active IL-18.

Interleukin-18 (IL-18) is a pro-inflammatory cytokine that evokes both innate and acquired immune responses. IL-18 is initially synthesized as an inactive precursor and the cleavage for processing into a mature, active molecule is mediated by pro-inflammatory caspases following the activation of inflammasomes. Two types of monoclonal antibodies were raised: anti-IL-1863-68 antibodies which recognize full-length1-193 and cleaved IL-18; and anti-IL-18 neoepitope antibodies which specifically recognize the new N-terminal 37YFGKLESK44 of IL-18 cleaved by pro-inflammatory caspase-1/4. These mAbs were suitable for Western blotting, capillary Western immunoassay (WES), immunofluorescence, immunoprecipitation, and function-blocking assays. WES analysis of these mAbs allowed visualization of the IL-18 bands and provided a molecular weight corresponding to the pro-inflammatory caspase-1/4 cleaved, active form IL-1837-193, and not to the inactive precursor IL-18, in the serum of patients with adult-onset Still's disease (6/14, 42%) and hemophagocytic activation syndrome (2/6, 33%). These monoclonal antibodies will be very useful in IL-18 and inflammasome biology and for diagnostic and therapeutic strategies for inflammatory diseases.

Possible role of p53/Mieap-regulated mitochondrial quality control as a tumor suppressor in human breast cancer.

Mitochondria-eating protein (Mieap), encoded by a p53-target gene, plays an important role in mitochondrial quality control (MQC). Mieap has been reported to have a critical role in tumor suppression in colorectal cancer. Here, we investigated its role as a tumor suppressor in breast cancer. The enforced expression of exogenous Mieap in breast cancer cells induced caspase-dependent apoptosis, with activation of both caspase-3/7 and caspase-9. Immunohistochemistry revealed endogenous Mieap in the cytoplasm in 24/75 (32%) invasive ductal carcinomas (IDC), 15/27 (55.6%) cases of ductal carcinoma in situ (DCIS) and 16/18 (88.9%) fibroadenomas (FA) (IDC vs DCIS; P = 0.0389, DCIS vs FA; P = 0.0234, IDC vs FA; P < 0.0001). In IDC, the Mieap promoter was methylated in 6/46 (13%) cases, whereas p53 was mutated in 6/46 (13%) cases. Therefore, the p53/Mieap-regulated MQC pathway was inactivated in 12/46 IDC (26.1%). Interestingly, all tumors derived from the 12 patients with Mieap promoter methylation or p53 mutations pathologically exhibited more aggressive and malignant breast cancer phenotypes. Impairment of p53/Mieap-regulated MQC pathway resulted in significantly shorter disease-free survival (DFS) (P = 0.021), although p53 status is more prognostic in DFS than Mieap promoter methylation. These results indicate that p53/Mieap-regulated MQC has a critical role in tumor suppression in breast cancer, possibly in part through mitochondrial apoptotic pathway.

Generation of the novel anti-FXYD5 monoclonal antibody and its application to the diagnosis of pancreatic and lung cancer.

Despite recent advances in cancer treatments, pancreatic cancer has a dismal prognosis globally. Early detection of cancer cells and effective treatments for recalcitrant tumors are required, but the innovative therapeutic tools remain in development. Cancer-specific antigens expressed only on cancer cells may help resolve these problems, and antibodies to such antigens have potential in basic research and clinical applications. To generate specific antibodies that bind to proteins expressed on the surface of pancreatic cancer cells, we immunized mice with human pancreatic cancer MIA PaCa-2 cells, and isolated a hybridoma that produces a monoclonal antibody (mAb), named 12-13.8. This antibody was applied to molecular biological experiments such as immunocytochemistry, immunoblotting, flow cytometry, and immunoprecipitation. In addition, we showed that mAb 12-13.8 could accumulate in tumors, through in vivo experiments using cancer-bearing mice. Immunohistochemical staining of pancreatic and lung tumor tissues indicated that the increase of the staining strength by mAb 12-13.8 positively and inversely correlated with the patients' cancer recurrence and survival rate, respectively. We identified the FXYD5 protein as the target protein of mAb 12-13.8, by a human protein array screening system. The FXYD5 protein is overexpressed in various types of cancer and is modified by O-linked glycosylation. We confirmed the binding of the FXYD5 protein to mAb 12-13.8 by using FXYD5-knockout MIA PaCa-2 cells, and detailed epitope mapping identified amino acid residues 45-52 as the minimal peptide sequence. Our results indicate that mAb 12-13.8 could be a valuable tool for FXYD5 studies, and useful in diagnostic and drug delivery applications for cancer patients.

The nuclear receptor constitutive active/androstane receptor arrests DNA-damaged human hepatocellular carcinoma Huh7 cells at the G2/M phase.

Here, we have demonstrated that xenobiotic activation of the nuclear receptor (CAR, NR1I3) can result in arresting DNA-damaged human hepatocellular carcinoma Huh7 cells at the G2/M phase. Huh7 cells over-expressing CAR were either treated with dimethyl sulfoxide, the CAR activator TCPOBOP (1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; androstenol, 16,(5α)-androsten-3α-OL), or repressor androstenol; these treatments were then followed by adriamycin treatment to damage DNA. FACS analysis revealed that CAR-activation by TCPOBOP increased the rate of arrested Huh7 cells at the G2/M phase (4N DNA content) after DNA damage by adriamycin. This increase correlated with the increase of cell viability in TCPOBOP-treated Huh7 cells, as determined by MTT assays. Real-time polymerase chain reaction analysis determined that, as regulated by CAR, the growth arrest and DNA damage-inducible γ (GADD45γ) and Cyclin G2 genes increased and decreased, respectively, as TCPOBOP increased the number of Huh7 cells arrested at the G2/M phase. Thus, the results suggest that CAR regulates cell cycle, increasing G2/M arrest, and delaying the death of DNA-damaged cells.

Kinetics of Anti-SARS-CoV-2 Antibody Response Following Two Doses of the BNT162b2 mRNA Vaccine: A Japanese Single-Center Primary Care Clinic Report Involving Volunteers and Patients with Autoimmune Disease.

Despite the promising effectiveness of the coronavirus disease 2019 vaccination using an mRNA vaccine, the short efficacy duration and some poor responses to the vaccination remain major concerns. We aimed to clarify the monthly kinetics of the anti-SARS-CoV-2 spike receptor-binding domain antibody response after two doses of the BNT162b2 vaccine in a Japanese population. A chemiluminescent enzyme immunoassay (CLIA) and an enzyme-linked immunosorbent assay were used to measure the antibody levels in 81 Japanese adults (age, <65 years). The antibody levels increased 10-fold at 2−3 weeks following the second dose of BNT162b2 and declined thereafter to approximately 50%, 20%, and 10% of the peak levels at 2, 3, and 6 months, respectively. To compare the antibody titers among different groups, older adults (age, >65 years; n = 38) and patients with systemic lupus erythematosus (SLE, n = 14) were also investigated. A decline in the mean relative antibody titers was observed in older men compared with younger men and in patients with SLE compared with individuals aged <65 years. Although the antibody levels increased drastically following two BNT162b2 doses, they then declined rapidly. Furthermore, poor responders to the vaccination were observed. Repeated vaccinations are required to maintain high antibody levels.

The nuclear receptors constitutive active/androstane receptor and pregnane x receptor activate the Cyp2c55 gene in mouse liver.

Mouse CYP2C55 has been characterized as an enzyme that catalyzes synthesis of 19-hydroxyeicosatetraenoic acid (19-HETE), an arachidonic acid metabolite known to have important physiological functions such as regulation of renal vascular tone and ion transport. We have now found that CYP2C55 is induced by phenobarbital (PB) and pregnenolone 16alpha-carbonitrile (PCN) in both mouse kidney and liver. The nuclear xenobiotic receptors constitutive active/androstane receptor (CAR) and pregnane X receptor (PXR) regulate these drug inductions: CYP2C55 mRNA was increased 25-fold in PB-treated Car(+/+) but not in Car(-/-) mice and was induced in Pxr(+/+) but not Pxr(-/-) mice after PCN treatment. Cell-based promoter analysis and gel shift assays identified the DNA sequence (-1679)TGAACCCAGTTGAACT(-1664) as a DR4 motif that regulates CAR- and PXR-mediated transcription of the Cyp2c55 gene. Chronic PB treatment increased hepatic microsomal CYP2C55 protein and serum 19-HETE levels. These findings indicate that CAR and PXR may play a role in regulation of drug-induced synthesis of 19-HETE in the mouse.

Possible role of semaphorin 3F, a candidate tumor suppressor gene at 3p21.3, in p53-regulated tumor angiogenesis suppression.

Although the regulation of tumor angiogenesis is believed to be one of the core functions of p53, the mechanism still remains to be elucidated. Here, we report that semaphorin 3F (SEMA3F), an axon guidance molecule, is involved in p53-regulated antiangiogenesis. The expression level of SEMA3F mRNA was increased by both exogenous and endogenous p53. Chromatin immunoprecipitation assay indicated that a potent p53-binding sequence in intron 1 of SEMA3F interacts with p53 and that it has a p53-responsive transcriptional activity. Overexpression of SEMA3F inhibited in vitro cell growth of the lung cancer cell line H1299. In nude mice assay, the size of the H1299 tumors expressing SEMA3F was much smaller, and they showed lesser number of blood vessels as compared with the control tumors. Moreover, tumors derived from the p53-knockdown colorectal cancer cell line LS174T displayed a remarkable enhancement of tumor vessel formation as compared with control tumors containing normal levels of p53. The expression levels of SEMA3F and neuropilin-2 (NRP2), the functional receptor for SEMA3F, in p53-knockdown LS174T tumors were lower than those in the control tumors. Adenovirus-mediated SEMA3F gene transfer induced the remarkable in vitro growth suppression of the stable transformant of H1299 cells, which express high levels of NRP2. These results suggest that p53 negatively regulates tumor vessel formation and cell growth via the SEMA3F-NRP2 pathway.

B-cell linker protein prevents aneuploidy by inhibiting cytokinesis.

Aneuploidy is a hallmark of human cancers. Although the maintenance of genomic integrity by p53 is important in preventing aneuploidy, its mechanism remains to be elucidated. Here we report evidence that B-cell linker protein (BLNK) mediates the inhibition of cytokinesis, which generates tetraploidy but prevents aneuploidy. We identified BLNK as a transcriptional target of p53. Surprisingly, ectopic expression of exogenous BLNK inhibited cytokinesis, resulting in the formation of tetraploid cells. Indeed, BLNK was involved in the generation of spontaneously arising binucleate tetraploid cells. Interestingly, cytokinesis after DNA damage was inhibited in p21(-/-) and p53+/+ cells, but not in p53(-/-) cells. BLNK knockdown in p53+/+ and p21(-/-) cells enhanced cytokinesis after DNA damage, leading to the generation of aneuploid cells. In addition, a BLNK-downregulated human pre-B leukemia cell line showed increased cytokinesis and aneuploidy after DNA damage compared with two other pre-B leukemia cell lines expressing higher levels of BLNK. These results suggest that BLNK acts as a mediator of p53 in the inhibition of cytokinesis, which prevents aneuploidy. We propose that the inhibition of cytokinesis is crucial for the maintenance of genomic integrity.

Precise epitope determination of the anti-vimentin monoclonal antibody V9.

Vimentin is a type III intermediate filament protein that is typically expressed in mesenchymal cells. Overexpression of vimentin is frequently observed in several types of cancer and is often associated with epithelial­to­mesenchymal transition. It was recently reported that the serum vimentin level is significantly elevated in colon and liver tumors. Therefore, a more sensitive vimentin detection system may be useful for cancer screening and early detection. The V9 mouse monoclonal antibody (mAb), which recognizes the human vimentin protein, is widely used in routine pathology to identify mesenchymal cells using immunohistochemical analysis. Although it has been suggested that the epitope of the V9 mAb is located within the C­terminal region of vimentin, the precise amino acid sequence that it recognizes has not yet been identified. In the present study, we constructed several deletion mutants of the vimentin protein and examined their reactivity with the V9 mAb to accurately map its epitope. We confirmed that its epitope resides in the C­terminal region of vimentin, between amino acids 392­466. Additionally, cross­species comparison of amino acid sequence alignment of vimentin, as well as site­directed mutagenesis, revealed that one residue, the asparagine at position 417, is critical for antibody binding. Using smaller vimentin fragments ranging in length from 9 to 13 residues, each containing this critical asparagine, we determined that the minimal residues required for V9 mAb recognition of human vimentin are the thirteen amino acid residues at positions 411-423 (411ISLPLPNFSSLNL423).

Identification of a

The introduction of a human chromosome 1 via microcell-mediated chromosome transfer (MMCT) induces the cellular senescence in mouse melanoma B16-F10 cells. The senescent cells maintained still the telomerase activity, which is frequently associated with immortal growth of human cells, suggesting that a telomerase-independent mechanism is involved in the senescence observed in this mouse cell line. To map the senescence-inducing gene to a specific chromosomal region, we took two experimental approaches: identification of a minimal region with the senescence-inducing activity via MMCT of a series of subchromosomal transferrable fragments (STFs), each consisting of a different profile of human chromosome 1-derived regions, and identification of a region commonly deleted from the transferred chromosome 1 in the revertant clones that escaped cellular senescence. These approaches identified a 2.7-3.0 Mb of senescence-inducing region shared among the active STFs and a 2.4-3.0 Mb of commonly deleted region in the revertant clones. These two regions overlapped each other to map the responsible gene at the 450 to 600-kb interval between UniSTS93710 and D1S3542 on chromosome 1q42.3. This study provides essential information and materials for cloning and characterization of a novel senescence-inducing gene that functions in a telomerase-independent pathway, which is likely to be conserved between mice and humans.

BNIP3 and NIX mediate Mieap-induced accumulation of lysosomal proteins within mitochondria.

Mieap, a p53-inducible protein, controls mitochondrial quality by repairing unhealthy mitochondria. During repair, Mieap induces the accumulation of intramitochondrial lysosomal proteins (designated MALM for Mieap-induced accumulation of lysosome-like organelles within mitochondria) by interacting with NIX, leading to the elimination of oxidized mitochondrial proteins. Here, we report that an additional mitochondrial outer membrane protein, BNIP3, is also involved in MALM. BNIP3 interacts with Mieap in a reactive oxygen species (ROS)-dependent manner via the BH3 domain of BNIP3 and the coiled-coil domains of Mieap. The knockdown of endogenous BNIP3 expression severely inhibited MALM. Although the overexpression of either BNIP3 or NIX did not cause a remarkable change in the mitochondrial membrane potential (MMP), the co-expression of all three exogenous proteins, Mieap, BNIP3 and NIX, caused a dramatic reduction in MMP, implying that the physical interaction of Mieap, BNIP3 and NIX at the mitochondrial outer membrane may regulate the opening of a pore in the mitochondrial double membrane. This effect was not related to cell death. These results suggest that two mitochondrial outer membrane proteins, BNIP3 and NIX, mediate MALM in order to maintain mitochondrial integrity. The physical interaction of Mieap, BNIP3 and NIX at the mitochondrial outer membrane may play a critical role in the translocation of lysosomal proteins from the cytoplasm to the mitochondrial matrix.

Identification of 14-3-3γ as a Mieap-interacting protein and its role in mitochondrial quality control.

Mieap, a p53-inducible protein, controls mitochondrial integrity by inducing the accumulation of lysosomal proteins within mitochondria. This phenomenon is designated MALM, for Mieap-induced accumulation of lysosome-like organelles within mitochondria. To identify this novel Mieap-interacting protein(s), we performed a two-dimensional image-converted analysis of liquid chromatography and mass spectrometry (2DICAL) on the proteins immunoprecipitated by an anti-Mieap antibody. We indentified 14-3-3γ as one of the proteins that was included in the Mieap-binding protein complex when MALM was induced. The interaction between Mieap and 14-3-3γ was confirmed on the exogenous and endogenous proteins. Interestingly, 14-3-3γ was localized within mitochondria when MALM occurred. A 14-3-3γ deficiency did not affect the accumulation of Mieap and lysosomal proteins within mitochondria, but dramatically inhibited the elimination of oxidized mitochondrial proteins. These results suggest that 14-3-3γ plays a critical role in eliminating oxidized mitochondrial proteins during the MALM process by interacting with Mieap within mitochondria.

Role of a novel CAR-induced gene, TUBA8, in hepatocellular carcinoma cell lines.

Phenobarbital (PB), a nongenotoxic carcinogen, activates the nuclear constitutive active/androstane receptor (CAR), resulting in the transcriptional induction or repression of various hepatic genes. We previously demonstrated that liver tumors developed after chronic PB treatment only when CAR is present. To understand the molecular mechanism of tumor promotion, cDNA microarray analysis was performed. We identified tubulin alpha 8 (TUBA8) as one of the candidate genes that may be involved in liver tumor promotion. Tuba8 mRNA was induced with PB treatment in mouse livers before tumor development as well as in tumor tissues. Because the functions of TUBA8 are unknown in liver, we investigated the effects of TUBA8 gene expression on cell growth, proliferation, and cell migration. Sense or antisense cDNA for Tuba8 was stably transfected into Huh7 and HepG2 cells. Exogenous overexpression of Tuba8 inhibited cell growth and proliferation in Huh7 but not in HepG2 cells, while cell migration was increased in HepG2 cells but not Huh7 cells. These results indicate that TUBA8 can play a role in the regulation of cell growth, proliferation, and cell migration in a cell-specific manner in vitro, suggesting that TUBA8 may contribute to mouse liver tumorigenesis through these functions.

Nuclear receptor CAR-regulated expression of the FAM84A gene during the development of mouse liver tumors.

The nuclear xenobiotic receptor CAR is a phenobarbital (PB)-activated transcription factor. Using a mouse model of two-step liver tumorigenesis, in which tumor growth was initiated by diethyl nitrosamine (DEN) and promoted by chronic treatment with PB, we previously demonstrated that tumors developed only in the presence of CAR. Here, we have identified the FAM84A (family with sequence similarity 84, member A) gene as a CAR-regulated gene that is over-expressed during development of phenobarbital-promoted mouse liver tumors. FAM84A mRNA was induced in the liver of DEN/PB-treated mice prior to the development of liver tumors and this induction continued in the non-tumor as well as tumor tissues of a tumor-bearing liver. Western blotting demonstated that FAM84A protein expression increased in mouse liver after PB treatment; however, the FAM84A protein in liver and liver tumors was not phosphorylated at the serine 38 residue, which has been reported to correlate with morphological changes in cells. Immunohistochemistry analysis revealed the cytoplasmic localization of FAM84A protein and its expression during tumor development in normal tissues (especially in hepatocytes around the central vein), eosinophilic foci, adenomas and carcinomas. HepG2 cell-based reporter assays indicated that CAR activated the FAM84A promoter. Exogenous over-expression of FAM84A in HepG2 cells resulted in increased cell migration. The physiological function of FAM84A remains unknown, but our results suggest that FAM84A is up-regulated by CAR during the development of liver tumors, and may play an important role in the progression of liver cancer by increasing cell migration.

Identification of NEEP21, encoding neuron-enriched endosomal protein of 21 kDa, as a transcriptional target of tumor suppressor p53.

NEEP21, also designated D4S234E or NSG1, is an endosomal protein expressed in neuronal cells under normal conditions. Here, we report that NEEP21 is a direct transcriptional target gene of the tumor suppressor p53. NEEP21 expression is inducible in non-neuronal human cancer cell lines by exposure to adriamycin, hydrogen peroxide, UV and γ-ray in a p53-dependent manner. Chromatin immunoprecipitation assay indicated that a potential p53-binding site (p53BS) is located in intron 1 of the NEEP21 gene. A reporter assay confirmed that p53BS has p53-responsive activity. The heterologous luciferase gene containing p53BS is also transactivated by p73-ß and p63-γ. The introduction of the NEEP21 gene into various cancer cell lines suppressed cell growth. Infection with an adenovirus vector containing NEEP21 induced apoptotic cell death via caspase-3 activation in many cancer cell lines. The expression of NEEP21 mRNA was remarkably induced by γ-ray irradiation in the spleen of p53+/+ mice but not in that of p53-/- mice. These results suggest that NEEP21 may play a critical role in apoptosis as a mediator of p53.

Regulation of apoptosis by p53-inducible transmembrane protein containing sushi domain.

The tumor suppressor p53 is a transcription factor that induces the transcription of various target genes in response to DNA damage and it protects the cells from malignant transformation. In this study, we performed cDNA microarray analysis and found that the transmembrane protein containing sushi domain (TMPS) gene, which encodes a putative type I transmembrane protein, is a novel p53-target gene. TMPS contains a sushi domain in the extracellular region, which is associated with protein-protein interaction. TMPS expression is induced by endogenous p53 under genotoxic stress in several cancer cell lines. Reporter assay revealed p53-dependent transactivation of the p53 binding-sites (BSs) located in the intron 1 of TMPS. Chromatin immunoprecipitation (ChIP) assay showed that p53 binds to these BSs in vivo. Overexpression of TMPS induced apoptosis through the activation of caspase-3, 8, and 9 in various cancer cell lines. Moreover, γ-irradiation induced the expression of TMPS mRNA in the spleen and colon of p53+/+ mice but not in those of p53-/- mice. These data indicate that TMPS may play a role in p53-dependent apoptosis under DNA damage condition.